This application concerns conferring nuclear male sterility to plants by transforming them with recombinant DNA and methods to restore fertility in these male sterile lines.
It has since long been acknowledged that seeds derived through cross-pollination between different parental lines of one species, give rise to offspring with better characteristics in terms of yield, uniformity, environmental fitness, and disease resistance, when compared with the offspring of seeds derived through self-pollination. This effect is generally referred to as the heterosis effect. For this reason it is an object for the seed industry to obtain hybrid seed in as many agricultural and horticultural crops as possible, because of their higher commercial value.
The development of hybrid cultivars of various plant species depends upon the capability of achieving almost complete crosspollination between parents. This is most simply achieved by rendering one of the parent lines male sterile (i.e. bringing them in a condition so that pollen is absent or nonfunctional) either manually, by removing the anthers, chemically or genetically by using, in the one parent, cytoplasmatic or nuclear genes that prevent anther and/or pollen development.
For hybrid plants where the seed is the harvested product (e.g. corn, wheat, oilseed rape) it is also necessary to ensure that fertility of the hybrid plants is restored. In systems in which the male sterility is under genetic control this requires the existence and use of genes that can restore male fertility. The development of hybrid cultivars by genetic modification is dependent on the availability of suitable and effective sterility and restorer genes.
Another goal of making plants male sterile is in protection of the (parent line) germplasm and prevention of breeding with farm-saved seeds and/or plants. If it is possible to create seeds resulting in homozygous sterile plants, it is impossible to obtain seed from them and to breed these further. Thus, a monopoly position for the distribution of these seeds or plants can be maintained. This is especially important for crops such as cauliflower, grasses, etc., in which the reproductive organs (as e.g. seed) are not the commercial target for the farmer. However, in order to propagate male sterile homozygous parent lines, the fertility should be restorable to obtain viable pollen.
Endogenous nuclear loci are known for most plant species that may contain genes which effect male fertility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant xe2x80x98male fertilexe2x80x99 allele at such loci results in male fertility.
In the last ten years it has been shown that a dominant male sterility trait can be induced in a plant by providing the genome of the plant with a recombinant DNA sequence coding, for example, for a cytotoxic product and under the control of a promoter which is predominantly active in selected tissue of the male reproductive organs.
In the prior art already a number of DNA sequences inducing male sterility and specific promoters have been elucidated.
In the International Patent Application WO 90/08830, ICI proposes methods for the production of restorable male-sterile plants in general terms, essentially comprising expression of a) either a gene encoding a protein inhibitor, or b) a so-called killer gene, which said genes are to be expressed in the male flowers, leading to cell death of the anthers and associated tissues. Exemplified killer genes are those which upon expression have an effect on mitochondrial metabolism.
In the International Patent Application WO 90/08831, ICI discloses the inhibition of cell-respiration and mitochondrial function by expression of a disrupter gene, eventually resulting in the death of the cells in which these genes are expressed. Preferred disrupter proteins are (a) the mammalian uncoupling protein (UCP); (b) a mutated form of the gene for the xcex2-1 subunit of FI-ATPase, such that the changes result in the disability of the subunits to assemble into a functional ATP-synthase; (c) a mutated, synthetic form of the oli I gene encoding subunit 9 of the OF-ATPase; (d) mutated forms of a mitochondrial transit peptide in order to disrupt protein transport to mitochondria; and (e) gene-constructs involving a fusion between the xcex2-subunit (ATPase) gene from yeast and the P-galactosidase gene from E. coli, resulting in expression of a disrupting fusion protein. Preferably such expression, according to the specification, should be regulated under the control of a tapetum- or pollen-specific promoter.
In the International Patent Application WO 89/10396, PGS proposes methods in general terms for obtaining male-sterile plants, by transforming the nuclear genome of the plant with a so-called xe2x80x9cmale-sterility DNA.xe2x80x9d The xe2x80x9cmale-sterility DNAxe2x80x9d comprises DNA that encodes an RNA or polypeptide capable of disturbing the proper metabolism, functioning, and/or development of any stamen cell in which it is expressed, preferably leading thereby to the death of any such stamen cell. Examples of such xe2x80x9cmale-sterility DNAsxe2x80x9d are those encoding DNAses, RNAses, proteases, or enzymes of phytohormone synthesis, such as cytokinin. Alternatively, it is proposed to select xe2x80x9cmale-sterility DNAsxe2x80x9d from antisense DNAs, xe2x80x9cwhich encode a strand of DNA complementary to a strand of DNA that is naturally transcribed in the plant""s stamen cells.xe2x80x9d
In the European Patent Application EP-A-0 329 308, Palladin Hybrids proposes a method to provide male-sterile plants, comprising producing a genetically transformed female parent, by essentially inserting into the genome of the said plant recombinant DNA sequences comprising antisense DNA, which blocks the production of functional pollen grains or renders the developing pollen grains susceptible to a chemical agent or physiological stress that blocks the production of functional pollen grains. Preferably, said antisense genes are expressed under the control of a pollen- specific promoter. Genes which are critical to production of functional pollen grains, according to the specification of EP-A-0 329 308, are to be selected from genes that are specifically expressed in the microspores, preferably in the premeiotic stage. Examples of microspore specific clones are LA and L19, derived from Brassica napus. Apart from the general indication to premeiotic genes and the expressly mentioned clones, no further teachings are given with respect to the nature of the genes the expression of which is to be blocked.
Also compounds of the flavonoid pathways have been used. EP 0 513 884 (MOGEN) is directed to the anther-specific disruption of the chalcone synthase pathway. Similarly, disruption of the anthocyanin biosynthesis in the seeds has been proposed (WO 95/34634, PGS).
Restoration of fertility has also been described. WO 94/09143 (MOGEN) mentions a restoration system for the anther-specific disruption of the chalcone synthase pathway. A sterility-fertility system has been provided in EP 0 628 635 (NUNHEMS), where sterility is caused by locally creating defects in amino acid biosynthesis pathways, while restoration is envisaged by supplying the missing amino acid or precursors through watering or spraying. WO 89/10396 (PGS) proposed to restore the fertility in plants made sterile by expression of the RNAse barnase by introducing a gene encoding barstar, the specific inhibitor of barnase.
Restoration by excising the gene responsible for the sterility through site-specific recombination by crossing a sterile plant with a plant expressing a recombinase has been disclosed in WO 97/13401 (Purdue Res. Found.).
However, there is still a need for a system that confers complete male sterility and is equally well subject to restoration.
This invention is directed to a method to make a plant male sterile by transforming it with a recombinant DNA capable of expression of a protein in the tapetum, pollen and/or anthers, characterized in that the protein is trehalose phosphate phosphatase (TPP). Said recombinant DNA comprising the gene coding for TPP is of bacterial, fungal, animal, plant or human origin, preferably derived from Escherichia coli.
Also part of the invention is a recombinant DNA comprising a tapetum, pollen and/or anther specific promoter and a gene coding for TPP, preferably a gene of bacterial, fungal, animal, plant or human origin, more preferably derived from Escherichia coli. Furthermore, vectors comprising this recombinant DNA and Agrobacterium strains comprising this vector form part of the invention. Equally plants transformed with this Agrobacterium strain or, in general, plants comprising the above-described recombinant DNA or plants made according to the above-described method form part of the invention.
Another embodiment of the invention is a method for making a plant male sterile and susceptible to restoration of male fertility by transforming a plant with recombinant DNA encoding TPP and which coding sequence is flanked by target sites of a site-specific recombinase. The accompanying method for restoration of fertility in a male sterile plant made according to this method is characterized in that the recombinant DNA encoding TPP is removed by providing said plant, either through transformation or through crossing, with a recombinant DNA capable of expressing a site-specific recombinase.
Also an embodiment of the invention is a method for making a plant male sterile and susceptible to restoration of male fertility by transforming it with a recombinant DNA capable of expression in the tapetum, pollen and/or anthers, characterized in that the recombinant DNA comprises a gene coding for trehalose phosphate phosphatase (TPP) and a gene coding for trehalose phosphate synthase (TPS) or other genes which inhibit the effect of TPP, such as antisense trehalase or antisense TPP, which last genes are under control of an inducible promoter. The accompanying method for restoration of fertility in a male sterile plant made according to this method is characterized in that TPS is expressed by induction of the inducible promoter.
A further embodiment of this invention is found in a method for making a plant male sterile and susceptible to restoration of male fertility by transforming it with a recombinant DNA capable of expression in the tapetum, pollen and/or anthers, characterized in that the recombinant DNA comprises a gene coding for trehalose phosphate phosphatase (TPP) of which the expression is controlled by a suppressor molecule encoded by a gene which is controlled by an inducible promoter. The accompanying method for restoration of fertility in a male sterile plant made according to this method is characterized in that the suppressor is expressed by induction of the inducible promoter.
A further embodiment of the invention is a method of restoration of plants made male sterile by the expression of trehalose phosphate phosphatase by applying a solution of gibberellic acid to said plants and more especially to the flower buds.
Other embodiments of this invention are male sterile plants produced by the above-described methods and the corresponding male fertility-restored plants produced by the accompanying fertility restoration methods.
Also included in this invention are methods for producing a male sterile homozygous line by crossing a male sterile plant as obtained according to the methods of the invention with a restored male fertile plant also obtained through the methods of the invention. Also the male sterile homozygous line produced by this method forms part of the invention.
A final embodiment of the invention is a method for the production of fertile hybrid plants by
a. transforming a plant with a recombinant DNA capable of expressing a site-specific recombinase; and
b. crossing said plant with a male sterile plant produced by any of the above described methods.
Also, the fertile hybrid plants produced by this method are part of the invention.